There are several aspects of human brain tumor gene therapy, which are poorly understood in part due to the lack of efficient tools to visualize biological processes in vivo. For example, it is difficult to image immune response during viral oncolysis (Project 1) and to determine the long-term fate of genetically engineered Tcell or progenitor cells (Project 3). The overall goal of this Project is to develop and optimize in vivo imaging techniques that can ultimately be used to objectively monitor vector and cell based glioma therapies. The maging strategies utilized in this Project are based on previously developed agents (e.g. targeted CLIO, activatable NIRF) adapted to high throughput screening for rapid development. In the first aims, we will develop injectable cell-specific imaging agents to image native immune cells (CD4, CDS, NK, macrophages) in their micro-environments using phage and library screens. In parallel we will develop high-efficiency multilabel cell trackers to follow ex vivo labeled cells by different imaging modalities simultaneously. In the second aim we shall develop and validate novel imaging agents that are predictive of the efficacy of oncolytic antiglioma therapies, in particular targets (e.g. aminopeptidase N) that will emerge through rigorous screens. The goal of the third aim is to develop and validate novel constructs to not only follow neural progenitor cells in vivo but image their differentiation into terminal cells as a function of time, delivery routes, and microenvironments. Data from these studies should be highly useful to rapidly evaluate the therapeutic efficacy of cell-based therapies and to develop more efficient approaches. There is a high degree of interaction with other Projects and Cores.